PLANT PHYSIOLOGY , Vol 114, Issue 4 1255-1265, Copyright © 1997 by American Society of Plant Biologists
|
BIOCHEMISTRY AND ENZYMOLOGY |
Activation of Pollen Tube Callose Synthase by Detergents (Evidence for Different Mechanisms of Action)
H. Li, A. Bacic and S. M. Read
Plant Cell Biology Research Centre, School of Botany, University of Melbourne, Parkville, Victoria 3052, Australia (H.L., A.B., S.M.R.)
In pollen tubes of Nicotiana alata, a membrane-bound, Ca2+-independent
callose synthase (CalS) is responsible for the biosynthesis of the
(1,3)-[beta]-glucan backbone of callose, the main cell wall component.
Digitonin increases CalS activity 3- to 4-fold over a wide range of
concentrations, increasing the maximum initial velocity without altering
the Michaelis constant for UDP-glucose. The CalS activity that requires
digitonin for assay (the latent CalS activity) is not inhibited bythe
membrane-impermeant, active-site-directed reagent UDP-pyridoxal when the
reaction is conducted in the absence of digitonin. This is consistent with
digitonin increasing CalS activity bythe permeabilization of membrane
vesicles. A second group of detergents, including
3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonate (CHAPS),
Zwittergent 3-16, and 1-[alpha]-lysolecithin, activate pollen tube CalS 10-
to 15-fold, but only over a narrow range of concentrations just below their
respective critical micellar concentrations. This activation could not be
attributed to any particular chemical feature of these detergents. CHAPS
increases maximum initial velocity and decreases the Michaelis constant for
UDP-glucose and activates CalS even in the presence of permeabilizing
concentrations of digitonin. Inhibition studies with UDP-pyridoxal indicate
that activation by CHAPS occurs by recruitment of previously inactive CalS
molecules to the pool of active enzyme. The activation of pollen tube CalS
by these detergents therefore resembles activation of the enzyme by
trypsin.
